1. Field of the Invention
The present invention relates to a rapid method for cancer diagnosis which is suitable to detect the presence of a carcinogenic process in a subject in the asymptomatic phase or the early phase. The present invention further relates to an activator compound of carbonic anhydrase II, i.e., a tumor marker, which is present in the serum of subjects having cancer; to a method for treating cancer, which method comprises detecting cancer in the patient in the asymptomatic phase or in early stage by said method for cancer diagnosis; and to a kit for performing said method for cancer diagnosis. The method for cancer diagnosis of the invention is based on an effect of cancer patients serum on the activity of purified carbonic anhydrase II. It was found that the activity of purified carbonic anhydrase II is significantly increased by the addition of a suitable amount of the serum of cancer patients. On the other hand, neither the serum of healthy volunteers nor the serum of patients having other diseases except cancer show a similar effect.
2. Description of the Related Art
Carbonic anhydrase is a zinc-enzyme discovered by Meldrum et al. in 1932 (Meldrum N. U., Roughton J. W.: Carbonic Anhydrase: Its preparation and properties, J. Physiol. (London), 1933, 80, 113-142). This enzyme is located in red blood cells and in other cells of the organism participates in the maintenance of the acid-base equilibrium (Maren T. H.: Carbonic Anhydrase, Physiological Reviews 1967, 47, 585-782; Hewett-Emmett D., Hopkins P. J., Tashian R. E., Czelusniak J. Origins and molecular evolution of the carbonic anhydrase isozymes, Ann. N.Y. Acad. Sci. 1984, 429, 338-358). Eight distinct isozymes of carbonic anhydrase are known in vertebrates (designated from I to VIII), which have different physiological functions and are involved in: gastric acid secretion, aqueous humour and cephalo-rachidian fluid formation, pancreatic bicarbonat secretion, intermediary metabolism, calcification, etc.
Applicants' previous research work demonstrated that carbonic anhydrase specific inhibitors (acetazolamide, ethoxzolamide, benzthiazol-2-sulphonamide) administered orally in the usual therapeutical doses reduce gastric acid secretion and lead to disappearance of ulcer pain and to endoscopic healing of gastroduodenal ulcers within a very short time (I. Puscas et al.: Les inhibiteurs de I'anhydrase carbonique dans le traitement de I'ulcere gastrique et duodenal, Archive Frangaises des Maladies de I'Appareil Digestif, Paris, 1976, 65:577-583; I. Puscas: Treatment of gastroduodenal ulcers with carbonic anhydrase inhibitors, Ann. New York Acad. of Sciences, 1984, 587-591; I. Puscas: Carbonic anhydrase inhibitors in the treatment of gastric and duodenal ulcers, in: New Pharmacology of Ulcer Disease, S. Szabo, Gy. Mozsik (Editors) Elsevier Publ. House, New York, USA,164-178, 1987; I. Puscas: Farmacologia Clinica da Ulcera Peptica, In: Aparelho Digestivo, Clinica e Cirurgia, Julio Coelho (Ed) MEDSI Publ. House, Rio de Janeiro, The 1st Edition in 1990,The 2nd Edition in 1996,1704-1734; I. Puscas: Avaliacao do paciente com doenca do estomago e duodeno, In: Aparelho Digestivo. Clinica e Cirurgia, Julio Coelho(Ed.), MEDSI Publ. House, Rio de Janeiro, Brasil, The 1st Edition in 1990, The 2nd Edition in 1996, 173-179).
Further research of applicants has shown that histamine, gastrin and acetylcholine--major stimulators of gastric acid secretion--directly activate carbonic anhydrase II and IV in gastric mucosa (I. Puscas: New concepts concerning the mechanism of stimulation of gastric acid secretion by histamine. VI.sup.th World Congress of Gastroenterology, Madrid, 1978, 213; I. Puscas: Direct activation by histamine of the carbonic anhydrase in the human gastric mucosa, Rev. Roum. Biochim.,1979, 4:317-320). Applicants' studies have also proved that the isozyme I of carbonic anhydrase which is located in red blood cells and in vascular walls is involved in the processes of vascular modulation (I. Puscas et al.: Inhibition of carbonic anhydrase by nitric oxide. Arzneimittel--Forschung/Drug Research, 1995, 8: 846-848; I. Puscas et al.: Prostaglandins having vasodilating effects inhibit carbonic anhydrase while leukotriens B.sub.4 and C.sub.4 increase carbonic anhydrase activity. Int. J. Clin. Pharmacol Therapeutics, 1995, 32, 3: 176-181; I. Puscas et al.: Isosorbide nitrates, nitroglycerine and sodium nitroprusside induce vasodilation, concomitantly inhibiting down to abolishment carbonic anhydrase I in erythrocytes. American Journal of Hypertension, 1997, 10 (1), 124-128), The izozyme II of carbonic anhydrase--located in the red blood cells and in the secretory cells--modulates the secretory processes in the organism (I. Puscas et al.: World Congress of Gastroenterology, Los Angeles, October 1994: 1329-32; 1366-81; I. Puscas et al.: Nonsteroidal anti-inflammatory drugs activate carbonic anhydrase by a direct mechanism of action. J. Pharmacol. Exp. Therap., USA, 1996,277, 3:1146-1148).
Applicants' results lead to postulation of a new theory for the signal transmission within the cell: The pH Theory. According to this theory, the stimuli or the primary messengers which produce vasculary and secretory modifications act by a dual mechanism: both on cellular membrane specific receptors and directly on bound-membrane carbonic anhydrase. This enzyme by its activation or inhibition assures an adequate pH for the stimulus-receptor complex formation in order to transmit the information to cellular effector. By this way through the pH modifications initiated by carbonic anhydrase this enzyme is involved in the modulation of physiological and pathological processes in the organism as well as in carcinogenesis (I. Puscas et al.: Carbonic anhydrase and modulation of physiologic and pathologic processes in the organism, I. Puscas--(Editor), Helicon Publishing House Timisoara, Romania, 1994, the Romanian version p.155-205 and 577-585, and the English version p. 147-197 and 551-559; I. Puscas: Carbonic anhydrase--a modulator of physiologic and pathologic processes in the organism: The pH Theory. The 4th International Conference on the Carbonic Anhydrases, July 1995, University of Oxford, England; I. Puscas: Carbonic anhydrase modulating physiological and pathological processes in organism. The pH theory. The Medical Novelty (Noutatea Medicala), Bucharest, Romania, 1997, 3:5-15)
The past few years have witnessed remarcable progress in understanding the biologic and biochemical bases for cancer. Cancer is a disease characterized by the uncontrolled growth of abnormal cells that spread from the anatomic site of origin to other tissues. This metastatic process involves a variety of cellular events taking place before any tumor cell gives rise to a metastatic colony. This spread, if uncontrolled, is the primary cause for the fatal outcome of cancer diseases. However, many cancers can be cured if they are detected early and treated promptly; others can be controlled for many years with a variety of treatment approaches.
Applicants' in vitro and in vivo studies have proved that carcinogenic substances reduce carbonic anhydrase II and superoxide dismutase activity and anticarcinogenic ones increase these enzymes' activity. (I. Puscas et al.: Relation between carbonic anhydrase I, CA II, non-steroidal anti-inflammatory drugs and some carcinogenic substances, Digestive Disease Week, San Diego, Calif., May 1995, 0643; I. Puscas et al.: The values of superoxid dismutase in patients with gastric cancer as compared to controls, Digestive Disease Week, San Francisco, Calif., May 1996, 0798; I. Puscas et al.: Anticancer chemotherapy increases red cell and gastric mucosa carbonic anhydrase activity. In vivo studies. Digestive Disease Week, San Francisco, Calif., May 1996, 0797). Other recent studies of applicants--performed with patients having different forms of cancer (histologically and computed tomography confirmed)--have shown that red blood cell carbonic anhydrase II and superoxide dismutase activity are weak in all patients, as compared to control group constituted by healthy volunteers (I. Puscas et al.: The effect of benzpyrene and cyclophosphamide on superoxid dismutase and carbonic anhydrase activity in gastric carcinoma patients as compared to controls. The First Forum of Gastroenterology, "Banat-Crisana", Timisoara, Romania, October 1996).
Up to the present, in many cases early detection of tumors and careful monitoring of the disease is conceivable by using tumor markers which are the biochermical indicators for the presence of a neoplastic proliferation and that can be detected in serum, plasma, or other body fluids. The main utility of tumor markers such as carcinoembryonic antigen (CEA), tumor necrosis factor (TNF-.alpha. and .beta.), sialic acid, prostate specific antigen (PSA), etc., has been in determining the response to therapy, the detection of residual disease or relapse. Until today no tumor marker has shown to be specific; virtually most of them are present at low levels in the normal physiological state or in non-neoplastic disease. Thus, these markers are not applicable in a general screening test for cancer.
In order to eliminate the major disadvantages of tumoral markers this invention is concerned in establishing a rapid method of screening for cancer in its asymptomatic stage, or their confirmation in the symptomatical phase. This early detection of cancer disease allows an efficient surgical or drug therapy along with other usual method utilized for tumor localization.
Applicant has now found that the activity of purified carbonic anhydrase II is significantly activated by the serum of cancer patients and that such activation is not observed with serum of healthy persons or serum of patients having other diseases but cancer. Starting from these observations, applicants found that by testing the effect of blood serum on the activity of purified carbonic anhydrase II is possible to diagnose any type of cancer, even those in early stages.